This invention relates to immunological control of fertility utilizing the antigenic, noncellular proteinaceous coating of the mammalian egg, the zona pellucida. The invention also relates to a water-soluble form of the zona pellucida; hereinafter referred to as "solubilized zona pellucida" or "SZP" for convenience. The invention further relates to vaccines comprising solubilized mammalian zona pellucida; to antisera comprising antibodies against the antigenic protein which constitutes the zona pellucida; to processes for the preparation of such SZP, vaccines and antisera; and to methods of treatment comprising administering such vaccines and antisera for fertility control in the mammalian female.
The possibility of utilizing immunological procedures for fertility control both in the male and in the female has been much discussed, but to date none has proved feasible. Such prior art attempts are characterized by an inability to isolate an antigenic material in pure form, which is a necessary condition for the production of tissue specific antibodies. Another shortcoming of such prior art attempts is that antisera substantially specific to a certain genital tissue are species specific, that is, such antisera give no evidence of immune reaction with the analogous tissue from distinct animal species; such results additionally characterize the species-specificity of prior art antigen preparations. The requirements imposed upon any immunological approach to fertility must include tissue specificity and cross-reaction (as opposed to species-specificity) because unless there is tissue specificity the immunological procedure could not be considered safe and without cross-reaction any such immunological procedure would be extremely limited since the antigenic source material would have to be obtained from the species to be rendered infertile --an impossibility for fertility control of the human female.
The possibility of immunological control of fertility employing the antigenic mammalian zona pellucida has been considered. This structure, the zona pellucida, and the phenomena associated with it, including the "zona reaction", have been much studied in reproductive biology with respect to certain sperm-egg interactions: approach, attachment and penetration. Approach of sperm to egg appears to be a random process without numerical limit. Attachment (sperm to zona pellucida) appears to be attributable to definite sperm receptor sites on the surface of the zona and in all likelihood is limited numerically by available area. Penetration, however, is normally limited to a single sperm. On penetration the receptor sites are deactivated (zona reaction). If the receptor sites or sites peripheral thereto are antigenic, the possibility of creating antibodies which mask or alter the zona pellucida and thus preclude attachment and penetration is an attractive, non-hormonal approach to fertility control. But to date such an approach has fallen to the above discussed two hurdles, namely: species-specificity and lack of tissue specificity. While Applicant is bound by no theory, it would appear that such prior attempts were unsuccessful principally because the precise antigenic material critical for practice of the present invention was not obtained in functional form by prior art procedures which typically comprise gross saline extraction of macerated whole ovarian tissue. Antisera prepared from such saline extracts, although rendered to some degree ovary-specific by adsorption of unwanted antibodies by contacting with diverse somatic tissues, invariably are found species-specific; further it is extremely doubtful that such prior art antisera are even capable of entering into an immune response with the critical antigenic sites of the zona pellucida for reasons which will be made evident below.
Unexpectedly it has been discovered that a tissue specific, non-species dependent (cross-reacting) anti-fertility vaccine can be prepared from the mammalian zona pellucida by a process which comprises heating either zonae pellucidae, per se, intact eggs or ovarian tissue comprising eggs in an appropriate medium such as water such that the proteinaceous zona pellucida is solubilized. Such aqueous solutions comprising solubilized zona pellucida may be evaporated to dryness to provide a form of the zona protein which is readily again solubilized in aqueous solution. Thus for purposes of the present invention the term "solubilized zona pellucida" (SZP) is defined to embrace mammalian zona pellucida protein -- dry or in solution -- which has been rendered water soluble according to the method of the present invention. Antibodies prepared from such solubilized zona pellucida are not species-specific and specifically react with the zona pellucida of the mammalian animal being treated for fertility control.
Thus, it is an object of the present invention to provide a vaccine for immunological fertility control of the female mammal which provides a reversible state of infertility without disruption of the normal estrus cycle. A further object of the present invention is to provide such a vaccine for immunological fertility control of the female mammal comprising solubilized mammalian zona pellucida (SZP) and to provide antisera comprising antibodies ("anti-SZP") against the antigenic mammalian zona pellucida for fertility control; wherein said vaccine and antisera are tissue specific and are active across mammalian species lines. Further objects of the present invention are to provide processes for the preparation of such vaccine, antisera (including SZP and anti-SZP, per se) and to provide methods of treatment comprising administering such vaccine and antisera for immunological control of fertility in the mammalian female.